Septic shock occurs in more than 750,000 patients each year in the U.S. nearly 215, 000 of whom die. Activation of macrophages (MPh) by microbial products such as Gram negative bacterial lipopolysaccharide (LPS) or Gram positive bacteria such as Staphylococcus aureus (Sa) or the Sa cell wall component lipoteichoic acid (LTA) lead to the systemic inflammatory response of sepsis. Toll-like receptors (TLR) are pathogen recognition receptors that are activated by LPS, Sa or LTA. Both LPS and Gram positive TLR ligands exhibit signaling convergence through novel Gi coupled pathways. LPS or Sa tolerance induced by pre-exposure to low concentrations of LPS or Sa increases resistance to LPS or Sa lethality and alters MPh signal transduction and mediator release. In contrast to tolerance, LPS induces priming responses to Sa in MPh. In both tolerant and primed MPh, Gi protein-coupled signaling pathways are altered. Our hypothesis is: Coupling of heterotrimeric Gi proteins to toll-like receptor signaling pathways regulate inflammatory cell activation and de-activation. Two interrelated specific aims will address this hypothesis: 1. Determine the mechanisms of Gi protein differential regulation of cellular inflammatory signaling to TLR ligands in vivo, and in vitro in primary cells and genetically engineered inflammatory cells, and 2. Determine the mechanisms responsible for alteration of Gi protein coupled signaling pathways in tolerance and priming responses to endotoxin and Gram positive TLR ligands. In Specific Aim 1, pharmacologic inhibition of Gi protein function and mice genetically depleted of Gai2 or Gai1 and Gai3 will be examined for in vivo responses to LPS and polymicrobial sepsis and in vitro responses to LPS, Sa or LTA-induced signaling pathways. TLR receptor and post-receptor protein coupling to Gi proteins will be analyzed by coimmunoprecipitation and surface plasmon resonance. Specific Aim 2 examines the effect of LPS, Sa and LTA-induced tolerance and priming changes in Gi protein function and coupled signaling pathways. We propose that b-arrestin proteins and the phosphatidylinositol (PI)3 kinase pathway function to modulate Gi signaling in tolerance. Gi regulatory proteins and the PI3 kinase pathway will be investigated with novel molecular approaches and with mice with genetic deficiencies of these proteins. Delineation of novel pathways of Gi protein signaling and molecular mechanisms responsible for tolerance and priming may provide innovative therapeutic approaches for sepsis.